Soil microbial community shifts explain habitat heterogeneity in two Haloxylon species from a nutrient perspective

Abstract Haloxylon ammodendron and Haloxylon persicum (as sister taxa) are dominant shrubs in the Gurbantunggut Desert. The former grows in inter‐dune lowlands while the latter in sand dunes. However, little information is available regarding the possible role of soil microorganisms in the habitat heterogeneity in the two Haloxylon species from a nutrient perspective. Rhizosphere is the interface of plant–microbe–soil interactions and fertile islands usually occur around the roots of desert shrubs. Given this, we applied quantitative real‐time PCR combined with MiSeq amplicon sequencing to compare their rhizosphere effects on microbial abundance and community structures at three soil depths (0–20, 20–40, and 40–60 cm). The rhizosphere effects on microbial activity (respiration) and soil properties had also been estimated. The rhizospheres of both shrubs exerted significant positive effects on microbial activity and abundance (e.g., eukarya, bacteria, and nitrogen‐fixing microbes). The rhizosphere effect of H. ammodendron on microbial activity and abundance of bacteria and nitrogen‐fixing microbes was greater than that of H. persicum. However, the fertile island effect of H. ammodendron was weaker than that of H. persicum. Moreover, there existed distinct differences in microbial community structure between the two rhizosphere soils. Soil available nitrogen, especially nitrate nitrogen, was shown to be a driver of microbial community differentiation among rhizosphere and non‐rhizosphere soils in the desert. In general, the rhizosphere of H. ammodendron recruited more copiotrophs (e.g., Firmicutes, Bacteroidetes, and Proteobacteria), nitrogen‐fixing microbes and ammonia‐oxidizing bacteria, and with stronger microbial activities. This helps it maintain a competitive advantage in relatively nutrient‐rich lowlands. Haloxylon persicum relied more on fungi, actinomycetes, archaea (including ammonia‐oxidizing archaea), and eukarya, with higher nutrient use efficiency, which help it adapt to the harsher dune crests. This study provides insights into the microbial mechanisms of habitat heterogeneity in two Haloxylon species in the poor desert soil.


| INTRODUC TI ON
In arid and semi-arid ecosystems, shrubs create high-nutrient patches and spatial variability of soil properties in the low-nutrient matrix, termed "fertile islands" (Cao et al., 2016;Diedhiou-Sall et al., 2013). By increasing soil moisture and protecting understory soils from the effects of high temperature, shrubs help soils retain nitrogen (N), increase soil organic matter, and create local microsites for microorganisms (MacMahon & Wagner, 1985). These environmental modifications improve soil microbial communities via increased biomass and activity (Jia et al., 2010). In turn, shrubs stabilize sand dunes by forming fertile islands, which are ecologically and economically important in desert ecosystems (Cao et al., 2016).
As a result, the spatial distribution of desert shrubs has a pronounced influence on the biogeochemical cycles of soil nutrients (Cross & Schlesinger, 1999).
The interactions between plants and microorganisms are essential for maintaining plant function and ecological niche (Díaz-Muñoz, 2017). Most desert microbial communities appear to be maintained primarily via abiotic processes (Cary et al., 2010), since deserts represent extreme environments that result in low microbial diversity (Neilson et al., 2012). However, perennial shrubs maintain a tight internal cycle in deserts, where they readily absorb, transport, and recycle nutrients (Soussi et al., 2016). The truth is that root-associated microbiota contribute to such adaptations (Mukhtar et al., 2021). The presence or emergence of desert plants, especially shrubs, will inevitably affect the diversity of soil microbial communities, due to the priming effect from the plant rhizosphere. This is demonstrated by the greater soil microbial activity (i.e., CO 2 efflux) in soils that contain plant materials than those in bare (nonplant) soils (Dijkstra et al., 2009). Recent studies have also shown that soil microbial diversity, rather than plant diversity, is a key limiting factor affecting ecosystem functioning and stability in high-aridity regions (Hu et al., 2021). Although the biomass of soil microorganisms in desert areas is small, they are still highly active and play a significant role in promoting plant health (Köberl et al., 2011). East, Central Asia, Afghanitsan, and Iran (Pyankov et al., 1999) and are the dominant perennial shrubs in the Gurbantunggut Desert, center of the Eurasian Continent. H. ammodendron grows at the bottom of desert sand dunes, while H. persicum mainly grows along dune crests. The species are thus largely spatially segregated within the same ecosystem, and both play an important role in maintaining ecological stability of the desert (Xu et al., 2007). These two Haloxylon species from arid regions are highly water limited, and as a result, the habitat differentiation between H. ammodendron and H. persicum is explored mainly from the aspect of plant-water relationships (Dai et al., 2015). The focus on water use, however, may mask the additionally important role of nutrient limitation in desert plants. Soil nutrients are also highly limiting in desert ecosystems, and thus likely have an important influence on plant species composition (Huang et al., 2018). But so far, knowledge of nutrient availability and plant-associated microbial communities in the desert soil remains fragmentary and scarce.
Rhizosphere, as the interface of plant-microbe-soil interactions, is crucial to the regulation of soil carbon and nitrogen biogeochemical cycles (Pathan et al., 2020). As a result, it is the most important area for plants to absorb soil nutrients during their growth and development. Rhizosphere effects in arid deserts are more significant than that in farmland and forest soils because of the lower nutrient content of the soil. Likewise, the nutrient interception in the rhizosphere is also stronger in desert soil (Huang et al., 2016). The characteristics of rhizosphere soils may thus be one of the most direct ways in which desert plants utilize nutrients and adapt to harsh environment (Cao et al., 2016;Shmueli et al., 2007). Therefore, comparing the rhizosphere effects of two Haloxylon species on soil chemical and microbial properties can better understand the possible role of soil microorganisms in their habitat heterogeneity from a nutrient perspective.
This study thus focuses on the rhizosphere soils (0-60 cm) of H. ammodendron and H. persicum and their corresponding soil environments in the Gurbantunggut Desert. Given that N is the most crucial limiting nutrient factor for plant growth in arid area (Huang et al., 2016), we applied quantitative real-time PCR (qPCR) to evaluate the variations in the abundance of bacteria, eukarya, archaea, and N-transforming microorganisms (N-fixing microbes, ammoniaoxidizing bacteria, and ammonia-oxidizing archaea), as affected by the habitat and rhizosphere. Meanwhile, the study also evaluated compositional shifts in bacterial and eukaryal communities using MiSeq amplicon sequencing. On this basis, we compared rhizosphere effects between these two shrubs in term of microbial activity (i.e., respiration), abundance and community structure, as well as for soil properties. The objective of this study was to detect the relationship between soil microbes and the two Haloxylon species in adjacent and distinct niches from a nutrient perspective. We hypothesized that (1) The rhizosphere priming effect of H. ammodendron and H. persicum on microbial respiration are different and is related to the recruitment of microorganisms to the rhizosphere and (2) microbial communities in rhizosphere soils of the two shrubs should be clearly differentiated, which corresponds to the soil environmental conditions, especially nutrient availability.

T A X O N O M Y C L A S S I F I C A T I O N
Applied ecology, Biodiversity ecology, Biogeochemistry, Biogeography, Botany, Environmental sustainability, Microbial ecology, Soil ecology 2 | MATERIAL S AND ME THODS

| Site description
The study was conducted within the vicinity (44°17′ N, 87°56′ E, and 475 m a.s.l.) of the Fukang Station of Desert Ecology, Chinese Academy of Sciences. The site lies along the southern edge of the Gurbantunggut Desert. This region is a temperate desert with an arid continental climate that has a cold winter and dry hot summers.
The annual mean temperature is 6.6°C. The annual mean precipitation is 160 mm, of which 25% is generally snowfall. Potential annual evaporation is 900 mm (Dai et al., 2015). Dendritic and longitudinal sand dunes characterize the whole landscape. The sand dunes reach a height of 5 to 12 m. The zonal soils are Torripsamments with loamy fine sand texture (81.7% sand, 16.8% silt, and 1.5% clay for the inter-dunes). Compared with inter-dunes, dune crests have higher sand content (>95%) and lower nutrient content (Dai et al., 2015;Xie et al., 2015). The desert area is either covered mainly with shrubs and semi-shrubs or bare soil. These plant communities are poor in species, and most of them were single-layer structure of low coverage (Xu et al., 2007). Although H. ammodendron and H. persicum grow in adjacent and distinct habitats (interdunes and dune crests) in the desert (Figure 1), they (as sister taxa) share some similarities in morphology, deep root system, tree life forms, and photosynthetic pathway of C4 (Pyankov et al., 1999;Xu et al., 2007;Zou et al., 2010). The densities of adult plants are 700 and 120 plants ha −1 in the habitats of H. ammodendron and H. persicum, respectively (Dai et al., 2015).
Snowfall events in the region occur primarily between January and March, with snow cover reaching a depth of 20-30 cm. The combined inputs of snow melt and rainfall lead to the highest soil moisture content in spring (from April to May), which provides plenty of water for the germination and growth of desert plants (Zhou et al., 2009). During this period, shallow soil water is the main source of water used by both H. ammodendron and H. persicum (Dai et al., 2015). Likewise, the rhizosphere priming effect (i.e., decomposition of organic matter by microorganisms) is also strongest during this stage (Warembourg & Estelrich, 2001). The groundwater table depth is more than 4 m. shrubs that absorb shallow soil moisture are mainly distributed at the first 60 cm depth of soil profile, and the sampling depth was set at 0-60 cm (Xu et al., 2007). Meanwhile, three rhizosphere soil samples around each individual shrub were collected randomly in each site. We selected three sampling depths: 0-20, 20-40, 40-60 cm, mainly based on the differences in moisture and compactness along the soil profile (0-60 cm) and the sampling depths in the previous study (Zou et al., 2010). Samples taken at the same depth for each individual shrub were mixed to obtain a representative soil sample.

| Soil sampling and chemical analysis
Meanwhile, three sampling points also were randomly picked out in the interplant (bulk soils) both for the dune crest site and inter-dune site.
Next, soil samples were divided into three parts after removal of visible plant root and organisms. One part was stored at 4°C to measure microbial activity within 2 weeks; one was frozen at −80°C for analysis of microbial gene sequences; the remaining samples were air-dried and passed through a 2-mm sieve for F I G U R E 1 Haloxylon ammodendron growing on interdune lowlands (a) and Haloxylon persicum growing on sand dunes (b) in the Gurbantunggut Desert, center of the Eurasian continent determination of soil chemical properties. Generally, the fertilizer island effect is characterized by higher organic matter, total N (TN), and available nutrients in soils beneath than outside of shrub canopy (Maestre et al., 2009;Rong et al., 2016). In this study, soil properties, including water content (SWC), pH, electrical conductivity (EC), organic carbon (SOC), TN, available N (AN), and available phosphorus (AP) were determined with the methods described by Li et al. (2019). Extractable ammonium and nitratenitrogen (NH 4 + -N and NO 3 − -N) contents were determined by the AA3 flow injection analyzers (FIA SFA CFA).

| Microbial activity, soil DNA extraction, and quantitative PCR
Soil respiration, which implies the intensity of SOC mineralization, was used as an indicator of microbial activity, and was measured using the alkali absorption method (Menyailo et al., 2003). After adjusting to 40% water-holding capacity, soil samples were conditioned at 25°C for 4 days in a thermostat-controlled container. Two beakers containing 10 ml of 1 M NaOH and deionized water were placed in the container to trap released CO 2 and keep the soil moist.
Total CO 2 efflux was measured using back-titration with a standardized 0.2 M HCL solution.
Soil DNA was isolated from a minimum of 0.5 g of dry weight soil (DWS) using a FastDNA spin kit (MP Biomedicals) and following the manufacturer's instructions. The integrity of the DNA extracts was confirmed by 0.8% agarose gel electrophoresis in 0.5TBE buffer. Moreover, the extracts were checked for quantity and quality using a Nanodrop ND-1000 UV-Vis Spectrophotometer (Nanodrop Technologies). By using quantitative real-time PCR (qPCR; Table 1), the target genes of bacteria, archaea, eukarya, ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and N-fixing microbes were analyzed to quantify their copy numbers. The recombinant plasmids were obtained from the positive clone with the correct DNA insert using a Miniprep kit (Qiagen). The plasmid concentration was determined by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). The standard curves of each gene were then obtained by diluting the recombinant plasmids with a 10-fold gradient. The melt-curve analysis at 65°C to 95°C (increment of 0.5°C) was performed to confirm PCR production specificity after amplification. Absolute copy number of the target gene was calculated from the plasmid DNA standard curve to generally estimate the abundance of specific microbial community (Okano et al., 2004).

| 16 S and 18 S rRNA gene pyrosequencing
The lichen soil crusts are common in the desert ecosystem (Huang et al., 2018). Lichens contain key members such as algae and microbes. As pioneers in desert and semi-desert ecosystems, they play a key role in the formation of desert soil and the colonization of vascular plants by increasing soil C, N, and P contents (Zhou et al., 2016).
Thus, while the soil bacterial community was analyzed, the eukaryal community was also analyzed to estimate changes in both fungal and algae communites in this study.
Accordingly, both 16 S and 18 S rRNA gene pyrosequencings were performed on a Miseq System sequencer (Illumia, Inc.). Briefly, DNA extracts from soil samples were used as templates for PCR amplification after quality control. Specific primers were tagged with unique, sample-specific barcodes before amplification. PCR was carried out with the 515F and 907R primers (Stubner, 2002) for the amplification of the V3-V4 region of the 16 S rRNA gene from bacteria and archaea.
PCR reactions were prepared in a 50 μl mixture containing 4 μl of dNTP Mixture (2.5 mM each), 1 μl of each primer at 10 μM, 1 μl of DNA and quantified with Picogreen (Invitrogen). The purified PCR amplicons with known concentrations were combined in equimolar ratios into a single tube in preparation for pyrosequencing analysis.
Next, the raw sequence reads were processed with the Quantitative Insights Into Microbial Ecology (QIIME version: 1.3.0) according to Caporaso et al. (2010). At the initial steps, the multiplexed reads (i.e., sequences obtained from DNA fragments) were assigned to samples by sample-specific tag sequences (barcodes).
Meanwhile, quality filtering of the reads was performed according to the characteristics of each sequence. After identifying and removing the impurities and low-quality sequences (quality score < 20), sequences >200 bp in length (quality score > 25), and without ambiguous base calls or mismatches were retained in sequencing analyses (Caporaso et al., 2010). These remaining high-quality sequences were then clustered into operational taxonomic units (OTU) using the threshold of 97% identity with the UCLUST algorithm (Edgar, 2010).
Meanwhile, the most abundant reads from each OTU were selected as the representative sequence of that OTU, which hence are more likely to be correct biological sequences. Taxonomic identity of the representative sequence from each OTU was determined using the Ribosomal Database Project (RDP) Classifier (http://rdp.cme.msu. edu/). The RDP Classifier assigns complete taxonomic information to each sequence in the database with 80% taxonomy confidence (Wang et al., 2007). Good's coverage, as an estimator of sampling completeness, was calculated at a 97% similarity cutoff (Claesson et al., 2009). The taxonomic assignments were used to construct an OTU table, which was a matrix of OTU abundance for each sample with specific taxonomic identifiers for each OTU (Suleiman et al., 2013). Meanwhile, the OTU sequence number was converted into the proportion of the OTU in the sample (i.e., the relative abundance) for cross comparison among the samples. A summary for pyrosequencing results targeting 16 S and 18 S rRNA genes in the sand dune site and inter-dune site was showed in Table 2.

| Data and statistical analyses
Rhizosphere effects on soil chemical and microbial properties were measured as the root soil (R/S) ratio. Statistical analyses were performed using SPSS 20.0 for Windows (SPSS Inc). Analysis of variance (ANOVA) and a Duncan's-test were used to assess significant differences (p < .05) in soil properties, microbial activity, gene copy number, and microbial relative abundances among rhizospheres of the two species and their corresponding bulk soils, after the data were checked for normality and homogeneity of variance. A twoway ANOVA was used to evaluate the effects of topography and rhizosphere on microbial activity and gene abundance. The Pearson correlation was used to test the significance of correlations between soil properties, microbial activity, and gene copy number.
Redundancy analysis (RDA) was performed to evaluate the relationship between soil properties and bacterial and fungal taxa. It was also used to fully assess changes in microbial community structure across the topography, rhizosphere, and soil depth. Species data

| Soil properties
The content of SOC and nutrient generally decreased with soil depth, except for the uniform distribution of NH 4 + -N throughout the soil profile (Table 3). Meanwhile, soil nutrient content of inter-dunes was substantially higher than that for dune crests

| Soil microbial activity and abundance
Soil microbial activity in the rhizosphere of both shrubs was greater than that found in bulk soils (p < .05; Figure 2   microbial activity was significantly stronger than that for H. persicum (p < .05), especially in topsoils.
The gene abundance of bacterial and archaeal 16 S rRNA and eukaryal 18 S rRNA substantially decreased with soil depth (Figure 3).

| Relative abundance of microbial taxa
For bacterial communities (Figure 5a), Actinobacteria and Proteobacteria were the predominant phyla in the desert soil.
Firmicutes and Chloroflexi were more abundant in inter-dune soils, For eukaryal communities, fungi were dominant members, especially on sand dunes (Figure 5b). Charophyta, Chlorophyta, and Ciliophora were more abundant in inter-dune soils, and Ochrophyta was more abundant in sand dune soils. Compared with bulk soils, the relative abundance of fungi and protozoa (e.g., Cercozoa and Ciliophora) was higher while microscopic algae were lower in rhizospheres of both shrubs (p < .05).

| Analyses of variance and correlation
Topography had a significant effect on microbial abundance (e.g., N-fixing microbes, bacteria, and archaea) and activity (p < .05), especially on the abundance of N-fixing microbes and bacteria and microbial activity (p < .001; Table 4). The plant rhizosphere also exerted a significant influence on microbial activity and abundance of eukarya, N-fixing microbes, AOB, archaea, and bacteria (p < .05). In addition, there were significant interactions between rhizosphere and topography on N-fixing microbe abundance and microbial activity (p < .01). In the desert, soil pH was positively correlated with the abundance of bacteria and N-fixing microbes (p < .05), while EC had no significant correlation with microbial abundance ( We found that most soil chemical properties (except AP, TN, and NH 4 + -N) significantly affected the soil microbial community in the desert (Table 6). The order of influence was AN > NO 3 − -N > SWC > SOC > pH > EC. The RDA ordination plot showed that the relationship between microbial communities and soil chemical properties in four sites (the bulk and rhizosphere soils of H. ammodendron on inter-dunes and H. persicum on sand crests; Figure 6). The rhizospheres at three depths (0-20, 20-40, and 40-60 cm) for H. ammodendron were centered on areas with relatively high moisture, SOC and N content, while the other sites (the rhizospheres of H. persicum and bulk soils for the two shrubs) were centered along a gradient with lower moisture, SOC, and N content.
Meanwhile, the topsoils of four sites were scattered in different areas in the ordination plot ( Figure 6). Also, the farthest distance was found between the two shrub rhizospheres. This showed that the microbial community structure between the two rhizospheres had the biggest difference in topsoils. However, the differences in In addition, some obligate bacteria can produce a range of bioactive compounds that can promote plant growth (Compant et al., 2010).
In this study, we found that Bacillus and Pseudomonas spp. were

| Variation of rhizosphere effects within two Haloxylon species
The current study confirms our hypothesis that there were significant variations in rhizosphere effects between the two Haloxylon species across different habitats. First, H. ammodendron showed a much stronger rhizosphere priming effect than did H. persicum ( Figure 2). In contrast, H. persicum had a more significant fertile island effect (higher R/S ratio of SOC and TN content) on the rhizosphere compared to H. ammodendron (Table 3). Moreover, the rhizosphere of H. ammodendron harbored more copiotrophs (e.g., Firmicutes, Bacteroidetes, and Proteobacteria; Griffith et al., 2017;Oh et al., 2012), N-fixing microbes and AOB, while the rhizosphere of H. persicum harbored more oligotrophs (e.g., actinomycetes; Zhang et al., 2021), archaea (Martens-Habbena et al., 2009), and fungi, which may be capable of growing as oligotrophs, chemolithoheterotrophs, or even as chemolithoautotrophs (Wainwright, 1988).
Meanwhile, this study also showed remarkable differences in microbial community structure between the rhizospheres of the two Haloxylon species. For example, the rhizosphere effect on the relative abundance of Bacillales and Pseudomonadales was stronger for H. ammodendron in inter-dunes than for H. persicum in dune crests.
As important plant growth-promoting bacteria, these two microbial groups can help root growth (van der Heijden & Schlaeppi, 2015).
In addition, the significant correlation between microbial activity and specific groups (e.g., N-fixing microbes and bacteria) demonstrates that soil priming effects are likely produced by rhizospherespecific microorganisms in the desert. These results suggest that SOC mineralization and nutrient consumption in the rhizosphere of H. ammodendron are stronger than that for H. persicum. Meanwhile, H. persicum may utilize soil nutrients more efficiently and economically, which would facilitate nutrient storage in the more hostile environment of dune crests. Moreover, such differences in nutrient utilization between the two Haloxylon species are closely related to the role of specific microorganisms in their respective rhizospheres.

| Factors driving variation in soil microbial communities
Topography significantly affects the distribution of soil water and nutrients, which in turn affects the distribution and growth of plants (Verma & Katpatal, 2021). Sand dunes and interdunes have a profound impact on the distributions of soil water and nutrients across the desert landscape by providing habitat heterogeneity (Dai et al., 2015). Due to stronger wind erosion and light radiation, soil nutrient and water conditions on sand dunes are significantly weaker than those observed on interdunes. Moreover, the plant rhizosphere has an extremely effective enriching effect and seletion effect on soil microbes via eutrophication and difference in soil characteristics caused by the terrain (Lugtenberg & Kamilova, 2009). Our study also confirms that microbial community structure across different ecological niches is the result of environmental filtering in topography and vegetation (Rewald et al., 2014).
Studies have shown that soil nutrients are the main driver of diversity and structural differentiation of soil microbes within the same soil type . Rhizosphere priming effects are primarily related to N availability, with P availability being less important (Sullivan & Hart, 2013). Our study also supports this finding and suggests the importance of N availability, particularly NO 3 − -N, for plant growth in desert ecosystems ( Figure 6 The N availability directly affects microbial activity due to the simultaneous assimilation of C and N by microbes (Chen et al., 2019).
The present study revealed positive correlations among nifH gene abundance, microbial activity, and soil AN (including NO 3 − -N) and SOC contents (Table 5 and Figure 6). This indicates that in the Gurbantunggut Desert, the rhizosphere of H. ammodendron on interdunes is closely related to soil N fixation and SOC mineralization, both of which are mediated mainly by soil bacteria. The rhizosphere of H. persicum attracted more fungi and actinomycetes than for H. ammodendron ( Figure 5). These microbes have higher nutrient use efficiency which likely helps H. persicum adapt to more stressful nutrient conditions (i.e., dune crests). Therefore, the assembly of microbial communities in the rhizosphere of the two Haloxylon species reflects the heterogeneity of their habitats by varied nutrient utilization strategies.

ACK N OWLED G M ENTS
We thank the anonymous reviewers for their valuable comments and suggestion on this manuscript; thanks also to all staff at the Fukang Station of Desert Ecology and Kang Jinhua for their assistance.

FU N D I N G I N FO R M ATI O N
This work was financially supported by the Special Project on

Regional Collaborative Innovation in Xinjiang Uygur Autonomous
Region, China (No.2022E01011) and the National Natural Sciences Foundation of China (Nos. 42271068, 32171874 and 42171068).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study will be openly avail-